Oligonucleotides and method for characterizing and detecting Genogroup II type small round structured virus

ABSTRACT

Nucleic acid sequences, oligonucleotides and a method for detection of SRSV, in particular, a virus which belongs to Genotype II (GII), in clinical examinations, public health examinations, food evaluations and food poisoning examinations are provided.

FIELD OF THE INVENTION

[0001] SRSV (Small Round Structured Virus) is commonly known as acausative virus of viral food poisoning. The present invention relatesto nucleic acid sequences, oligonucleotides and method for detection ofSRSV and, in particular, a virus which belongs to Genotype II (GII) inclinical examinations, public health examinations, food evaluations andfood poisoning examinations.

PRIOR ART

[0002] SRSV belongs to the human Calicivirus group. Human Calicivirusesare classified according to their three genetic types: Genogroup I (GI),Genogroup II (GII) and Genogroup III (GIII). Generally speaking, GI andGII Caliciviruses are generally referred to as SRSV, and GIIICaliciviruses are referred to as human Caliciviruses in the narrowsense.

[0003] Approximately 20% of the food poisoning cases reported in Japanare attributed to viral causes. SRSV is detected in over 80% of theseviral food poisoning cases. The major source of infection is food, andraw oysters are often implicated. SRSV has also been detected in infant(sporadic) acute enterogastritis, thus suggesting the possibility ofpropagation from human to human. SRSV detection therefore provides animportant contribution to public health and food quality.

[0004] To date, SRSV detection has been relied on electron microscopeobservation. Detection by this method, however, requires the virus to bepresent in an amount of 10⁶/ml or greater, and thus the detectionsubject was limited to patient's feces. Further, even though observationof the virus was possible, it could not be identified.

[0005] In recent years, it has become possible to produce viroid hollowparticles for human caliciviruses, and research is advancing toward aspecific antibody-detecting ELISA employing such particles. However, thedetection sensitivity is still on the same level as electron microscopy,and the method is therefore far from highly sensitive.

[0006] As mentioned above, since a complex procedure and a long time arerequired for the conventional method and it is difficult to detect traceamounts of SRSV in samples within a short time, it has been desired toprovide a detection method satisfying the high-speed andhigh-sensitivity requirements for food evaluation and the like. Therehas also been a demand for development of an automated examinationdevice which allows more convenient examination.

[0007] Methods of amplifying target nucleic acid can be utilized ashighly sensitive detection methods. One known method for amplificationof specific sequences of genomic RNA such as that of SRSV is the reversetranscription-polymerase chain reaction (RT-PCR). This method comprisessynthesis of a cDNA for the target RNA by a reverse transcription step,and then repeating a cycle of heat denaturation, primer annealing andextension reaction in the presence of a pair of primers which arecomplementary and homologous to both ends of specific sequences of thecDNA (the antisense primer may be the one used in the reversetranscription step) as well as a thermostable DNA polymerase, therebyamplifying the specific DNA sequence. However, the RT-PCR methodrequires a two-step procedure (a reverse transcription step and a PCRstep), as well as a procedure involving rapidly increasing anddecreasing the temperature, which prevent its automation.

[0008] Other methods known for amplification of specific RNA sequencesinclude the NASBA and 3SR methods which accomplish amplification ofspecific RNA sequences by the concerted action of reverse transcriptaseand RNA polymerase. In these methods, the target RNA is used as atemplate in the synthesis of a promoter sequence-containingdouble-stranded DNA using a promoter sequence-containing primer, reversetranscriptase and Ribonuclease H; this double-stranded DNA provides atemplate in the synthesis of an RNA containing the specific basesequence of the target RNA using an RNA polymerase; subsequently, thisRNA provides a template in a chain reaction for synthesizing adouble-stranded DNA containing the promoter sequence.

[0009] Thus, the NASBA and 3SR methods allow nucleic acid amplificationat a constant temperature and are therefore considered suitable forautomation. However, as these amplification methods involve relativelylow temperature reactions (41° C., for example), the target RNA forms anintramolecular structure which inhibits binding of the primer and mayreduce the reaction efficiency. Therefore, they require subjecting thetarget RNA to heat denaturation before the amplification reaction so asto destroy the intramolecular structure of the target RNA and thus toimprove the primer binding efficiency. Further, even when carrying outthe detection of an RNA at a lower temperature, these methods require anoligonucleotide capable of binding to the RNA forming such a molecularstructure.

[0010] Thus, an object of the present invention is to provide nucleicacid sequences, oligonucleotides or suitable combination thereof,capable of specifically cleaving or amplifying SRSV and, in particular,a virus which belongs to GII type, preferably at a relatively low andconstant temperature (between 35° C. and 50° C., preferably 41° C.),useful in detecting and identifying such a virus at high sensitivity.

DETAILED DESCRIPTION OF THE INVENTION

[0011] The invention of claim 1, which has been accomplished to achievethis object, relates to a cDNA as shown in SEQ. ID. No.1, or fragment orderivative thereof having a size sufficient to bind to Genogroup II typeSmall Round Structured Virus (SRSV).

[0012] The invention of claim 2, which has been accomplished to achievethe aforementioned object, relates to an oligonucleotide for detectionof GII type SRSV, which oligonucleotide is capable of binding to saidGII type SRSV at specific site, and comprises at least 10 contiguousbases of any of the sequences listed as SEQ. ID. Nos.2 to 9.

[0013] The invention of claim 3, which has been accomplished to achievethe aforementioned object, relates to the oligonucleotide according toclaim 2, wherein said oligonucleotide is an oligonucleotide probe forcleaving said RNA at said specific site by binding to said specific siteof said RNA.

[0014] The invention of claim 4, which has been accomplished to achievethe aforementioned object, relates to the oligonucleotide according toclaim 2, wherein said oligonucleotide is an oligonucleotide primer for aDNA elongation reaction.

[0015] The invention of claim 5, which has been accomplished to achievethe aforementioned object, relates to the oligonucleotide according toclaim 2, wherein said oligonucleotide is an oligonucleotide probe aportion of which is modified or labeled with a detectable marker.

[0016] The invention of claim 6, which has been accomplished to achievethe aforementioned object, relates to the oligonucleotide according toclaim 2, wherein said oligonucleotide is a synthetic oligonucleotide inwhich a portion of its base(s) is (are) modified without impairing thefunction of said oligonucleotide as an oligonucleotide probe.

[0017] The oligonucleotides of the present invention, which have beenaccomplished to achieve the aforementioned object, are oligonucleotidesthat complementarily bind in a specific manner to intramolecularstructure-free regions of the target RNA in the aforementioned RNAamplification, and they are capable of binding specifically to thetarget RNA without the heat denaturation described above. In thismanner, the present invention provides oligonucleotides that bind tointramolecular structure-free regions of the GII type SRSV RNA at arelatively low and constant temperature (35-50° C., and preferably 41°C.), which are useful for specific cleavage, amplification, detection orthe like of GII type SRSV RNA. More specifically, the present inventionrelates to an oligonucleotide primer which cleaves the target RNAmentioned above at specific site, an oligonucleotide primer foramplifying the above target DNA with PCR, an oligonucleotide primer foramplifying the above target DNA with NASBA or the like, and anoligonucleotide probe for detecting the target nucleic acid without orafter these amplifications, thereby accomplishes rapid and highlysensitive detection.

[0018] SEQ ID Nos. 2 through 9 illustrate examples of theoligonucleotides of the present invention useful in cleavage,amplification, detection or the like of RNA derived from GII type SRSV.In this connection, RNA derived from GII type SRSV also includes RNAthat has been produced by using these genes as templates. Although eachof the oligonucleotide of the present invention may include entire basesequence of any of SEQ ID Nos.2 to 9, since 10 contiguous bases areadequate for specific binding to GII type SRSV, these oligonucleotidescan be oligonucleotides comprising at least 10 contiguous bases of thedescribed sequences.

[0019] The oligonucleotides of the present invention can be, forexample, used as an RNA-cleavable probe. Cleavage of a target RNA at aspecific site can be accomplished by hybridizing the oligonucleotide ofthe present invention to a single-stranded target RNA, and then exposingit to an enzyme which cleaves only the RNA moieties of the heteronucleicdouble-stranded RNA-DNA. As for this enzyme, those which are known tohave common ribonuclease H activity can be used.

[0020] The oligonucleotides of the present invention can be used, forexample, as oligonucleotide primers for nucleic acid amplification. If anucleic acid amplification method is carried out using theoligonucleotide of the present invention as the primer, only the targetnucleic acid, namely nucleic acids of the GII type SRSV, can beamplified. Although examples of amplification methods include PCR, LCR,NASBA and 3SR, nucleic acid amplification methods that can be carriedout at a constant temperature such as LCR, NASBA and 3SR areparticularly preferable. GII type SRSV can be detected by detecting theamplification product by various methods. In this case, any of the aboveoligonucleotides other than the oligonucleotide used in theamplification may be used as probes, and the fragment of the amplifiedspecific sequence can be confirmed by electrophoresis or the like.

[0021] The oligonucleotides of the present invention can be used asprobes by, for example, modifying its portion or labeling it with adetectable marker. When detecting the target nucleic acid, theoligonucleotide of the present invention labeled with the detectablemarker may be hybridized to a single-stranded target nucleic acid, afterwhich the hybridized probe can be detected via the marker. The markerdetection may be carried out by a method suitable for the particularmarker and, for example, when using an intercalator fluorescent dye forlabeling the oligonucleotide, a dye with the property of exhibitingincreased fluorescent intensity by intercalation in the double-strandednucleic acid comprising the target nucleic acid, and the oligonucleotideprobe, may be used in order to allow easy detection of only thehybridized probe without removal of the probe that has not hybridized tothe target nucleic acid. When using a common fluorescent dye as themarker, the marker may be detected after removal of the probe that hasnot hybridized to the target nucleic acid. For the detection, the targetnucleic acid in the sample is preferably amplified to a detectableamount by a nucleic acid amplification method such as PCR, NASBA or 3SRmethod, among which isothermal nucleic acid amplification methods suchas the NASBA and 3SR methods are most preferable. When incorporating thenucleotide-labeled probe in the reaction solution during theamplification, it is especially preferable to modify the probe by, forexample, adding glycolic acid to the 3′-end so that the probe will notfunction as a nucleotide primer.

[0022] The invention of claim 7, which has been accomplished to achievethe aforementioned object, relates to a GII type SRSV RNA amplificationprocess in which the specific sequence of said GII type SRSV RNA presentin a sample is used as a template for synthesis of a cDNA employing anRNA-dependent DNA polymerase, the RNA of the formed RNA/DNA hybrid isdecomposed by Ribonuclease H to produce a single-stranded DNA, saidsingle-stranded DNA is then used as a template for production of adouble-stranded DNA having a promoter sequence capable of transcribingRNA comprising said specific sequence or the sequence complementary tosaid specific sequence employing a DNA-dependent DNA polymerase, saiddouble-stranded DNA produces an RNA transcription product in thepresence of an RNA polymerase, and said RNA transcription product isthen used as a template for cDNA synthesis employing said RNA-dependentDNA polymerase, wherein said RNA amplification process beingcharacterized by employing a first primer comprising at least 10contiguous bases, of any of the sequences listed as SEQ. ID. No.20 toNo.24, which has a sequence homologous to a portion of said GII typeSRSV RNA to be amplified, and a second primer comprising at least 10contiguous bases, of any of the sequences listed as SEQ. ID. No.25 toNo.31, which has a sequence complementary to a portion of said GII typeSRSV RNA sequence to be amplified (where either or both the first andsecond primers include the RNA polymerase promoter sequence at their 5′end).

[0023] The invention of claim 8, which has been accomplished to achievethe aforementioned object, relates to the process of claim 7, whereinsaid RNA amplification process is carried out in the presence of anoligonucleotide probe capable of specifically binding to the RNAtranscription product resulting from the amplification and labeled withan intercalator fluorescent pigment, and changes in the fluorescentproperties of the reaction solution are measured (with the proviso thatsaid labeled oligonucleotide is different from said firstoligonucleotide and said second oligonucleotide).

[0024] The invention of claim 9, which has been accomplished to achievethe aforementioned object, relates to the detection method of claim 8,characterized in that said probe is designed so as to complementarilybind with at least a portion of the sequence of the RNA transcriptionproduct, and the fluorescent property changes relative to that of asituation where a complex formation is absent.

[0025] The invention of claim 10, which has been accomplished to achievethe aforementioned object, relates to the detection method of claim 9,characterized in that said probe comprises at least 10 contiguous basesof any of the sequences listed as SEQ. ID. No. 32 to No. 35 or itscomplementary sequence.

[0026] The present invention provides a nucleic acid amplificationprocess for amplification of GII type SRSV RNA in a sample, and adetection method for RNA transcription products obtained by the nucleicacid amplification process. The amplification process of the inventionincludes the PCR, NASBA and 3SR methods, but is preferably a constanttemperature nucleic acid amplification method such as the NASBA or the3SR methods whereby GII type SRSV-specific RNA sequences are amplifiedby the concerted action of reverse transcriptase and RNA polymerase (areaction under conditions in which reverse transcriptase and RNApolymerase act in concert).

[0027] For example, the NASBA method is an RNA amplification process inwhich the specific sequence of GII type SRSV RNA present in a sample isused as a template for synthesis of a cDNA employing an RNA-dependentDNA polymerase, the RNA of the formed RNA/DNA hybrid is decomposed byRibonuclease H to produce a single-stranded DNA, the single-stranded DNAis then used as a template for production of a double-stranded DNAhaving a promoter sequence capable of transcribing RNA comprising thespecific sequence or the sequence complementary to the specific sequenceemploying a DNA-dependent DNA polymerase, the double-stranded DNAproduces an RNA transcription product in the presence of an RNApolymerase, and the RNA transcription product is then used as a templatefor cDNA synthesis employing the RNA-dependent DNA polymerase, and theprocess of the present invention is characterized by employing a firstprimer comprising at least 10 contiguous bases of any of the sequenceslisted as SEQ. ID. No. 20 to No. 24 which has a sequence homologous to aportion of the GII type SRSV RNA, and a second primer comprising atleast 10 contiguous bases of any of the sequences listed as SEQ. ID. No.25 to No. 31, which has a sequence complementary to a portion of the GIItype SRSV RNA sequence to be amplified (where either or both the firstand second primers include the RNA polymerase promoter sequence at their5′ region).

[0028] While there are no particular restrictions on the RNA-dependentDNA polymerase, the DNA-dependent DNA polymerase and the Ribonuclease H,AMV reverse transcriptase which has all of these types of activity ispreferred. The RNA polymerase is also not particularly restricted, butT7 phase RNA polymerase and SP6 phage RNA polymerase are preferred.

[0029] In this amplification process, there is added an oligonucleotidewhich is complementary to the region adjacent and overlapping with the5′ end of the specific sequence region (bases 1 to 10) of the GII typeSRSV RNA sequence, and the GII type SRSV RNA is cleaved (withRibonuclease H) at the 5′ end region of the specific sequence to preparethe initial template for nucleic acid amplification, thereby allowingamplification of GII type SRSV RNA without the specific sequence at the5′ end. The oligonucleotide used for this cleaving may, for example, beany of those of SEQ. ID. No. 25 to No. 31 (provided that it differs fromthe ones used as the first oligonucleotide in the amplificationprocess). The cleaving oligonucleotide is preferably chemically modified(for example, aminated) at the 3′ hydroxyl in order to prevent anextension reaction at the 3′ end.

[0030] The RNA amplification product obtained by the aforementionednucleic acid amplification process may be detected by a known detectionmethod but, preferably, the amplification process is carried out in thepresence of an oligonucleotide probe labeled with an intercalatorfluorescent pigment, while measuring the changes in the fluorescentproperties of the reaction solution. The oligonucleotide probe willtypically be the one wherein the intercalator fluorescent pigment isbonded to a phosphorus atom in the oligonucleotide by way of a linker.With this type of suitable probe, formation of a double strand with thetarget nucleic acid (complementary nucleic acid) causes the intercalatorportion to intercalate in the double-stranded portion resulting in achange in the fluorescent property, so that no separatory analysis isnecessary (Ishiguro, T. et al. (1996), Nucleic Acids Res. 24(24)4992-4997).

[0031] The probe sequence is not particularly restricted so long as ithas a sequence complementary to at least a portion of the RNAtranscription product, but it is preferably a sequence comprising atleast 10 contiguous bases of the sequences listed as SEQ. ID. Nos.32 toNo.35. Also, chemical modification (for example, glycolic acid addition)at the 3′ end hydroxyl group of the probe is preferred in order toprevent an extension reaction with the probe as a primer.

[0032] Accordingly, it is possible to amplify and detect RNA comprisingthe same sequence as the specific sequence of GII type SRSV RNA in asingle tube at a constant temperature and in a single step, thusfacilitating its application for automation.

BRIEF DESCRIPTION OF THE DRAWINGS

[0033]FIG. 1 is a urea modified 6% polyacrylamide electrophoresisdiagram for samples after performing GII type SRSV standard RNA bindingtests at 41° C., using oligonucleotides G2-1R to G2-17R (black and whiteinverted). The arrows indicate the positions of the specific bands.Lanes 1 to 17 show the results of the binding test using G2-1R to G2-17Rrespectively, and lane N represents the negative control (using only thediluent instead of RNA samples). The molecular weight markers (lanes M1and M2) used therein are RNA markers (0.1 to 1 kb and 0.2 to 10 kb).

[0034]FIG. 2 is a urea modified 6% polyacrylamide electrophoresisdiagram for samples after performing GII type SRSV standard RNA bindingtests at 41° C., using the oligonucleotides selected in Example 1 (blackand white inverted). The arrows indicate the positions of the specificbands. Lanes 1 to 8 show the results for GI type SRSV standard RNA ofthe binding tests, and lanes 9 to 16 show the results for GII type SRSVstandard RNA of the binding test. Lanes 1 and 9, lanes 2 and 10, lanes 3and 11, lanes 4 and 12, lanes 5 and 13, lanes 6 and 14, lanes 7 and 15,as well as lanes 8 and 16 used oligonucleotides G2-1R, G2-2R, G2-3R,G2-8R, G2-10R, G2-11R, G2-12R and G2-17R, respectively, and lane Nrepresents the negative control (using only the diluent instead of RNAsamples). The molecular weight markers (lanes M1 and M2) used thereinare RNA markers (0.1 to 1 kb and 0.2 to 10 kb).

[0035]FIG. 3 is an electrophoresis diagram for RNA amplificationreactions in Example 3 using oligonucleotide combinations (a) to (d)shown in Table 2 (black and white inverted), with an initial RNA amountof 10⁴ copies/test. Lanes 1 and 2 are the results for combination (a),lanes 4 and 5 are for combination (b), lanes 7 and 8 are for combination(c), lanes 10 and 11 are for combination (d), while lanes 3, 6, 9, and12 are for the negative control (using only the diluent instead of RNAsamples). The molecular marker used therein was φX174/Hae III digest(Marker 4). Specific bands were confirmed in every combination.

[0036]FIG. 4 is an electrophoresis diagram for RNA amplificationreactions in Example 3 using oligonucleotide combinations (e) to (h)shown in Table 2 (black and white inverted), with an initial RNA amountof 10⁴ copies/test. Lanes 1 and 2 are the results for combination (e),lanes 4 and 5 are for combination (f), lanes 7 and 8 are for combination(g), lanes 10 and 11 are for combination (h), while lanes 3, 6, 9, and12 are for the negative control (using only the diluent instead of RNAsamples). The molecular marker used therein was φX174/Hae III digest(Marker 4). Specific bands were confirmed in every combination.

[0037]FIG. 5 is an electrophoresis diagram for RNA amplificationreactions in Example 3 using oligonucleotide combinations (i) to (l)shown in Table 2 (black and white inverted), with an initial RNA amountof 10⁴ copies/test. Lanes 1 and 2 are the results for combination (i),lanes 4 and 5 are for combination (j), lanes 7 and 8 are for combination(k), lanes 10 and 11 are for combination (l), while lanes 3, 6, 9, and12 are for the negative control (using only the diluent instead of RNAsamples). The molecular marker used therein was φX174/Hae III digest(Marker 4). Of these combinations, specific bands were confirmed incombinations (k) and (l).

[0038]FIG. 6 shows a graph (A) of the fluorescence increase ratio whichincreases as the reaction time and production of RNA progress, and acalibration curve (B) obtained for the initial RNA amount logarithm andthe rising time, with an initial RNA amount of between 10¹ copies/testand 10⁵ copies/test in Example 4. The initial amount of 10³ copies/testof RNA was detectable after approximately 20 minutes of reaction, and acorrelation between initial RNA amount and rise time was demonstrated.

EXAMPLES

[0039] The present invention will now be explained in greater detail byway of examples, with the understanding that the invention is notlimited by these examples.

Example 1

[0040] Specific binding of the oligonucleotides of the invention to GIItype SRSV at 41° C. was examined.

[0041] (1) Of the GII type SRSV-RNA, a standard RNA (SEQ ID No.10)comprising a region of 2843 bases in total containing the SEQ ID No.1region and a portion of the structural protein-coding gene region, aswell as a 69 base-partial region derived from the 5′ end of a vector(pCR2.1, Invitrogen) was quantified by ultraviolet absorption at 260 nm,and then diluted to a concentration of 0.62 pmol/μl with an RNA diluent(10 mM Tris-HCl (pH 8.0)), 0.1 mM EDTA, 1 mM DTT, 0.5 U/μl RNaseInhibitor (Takara Shuzo Co. Ltd.).

[0042] (2) 14 μl of a reaction solution having the following compositionwas dispensed into 0.5 ml volume PCR tubes, (Gene Amp Thin-WalledReaction Tube™, Perkin-Elmer Co. Ltd.)

[0043] Reaction Solution Composition (each concentration represents thatin a final reaction solution volume of 15 μl)

[0044] 60 mM Tris-HCl buffer (pH 8.6)

[0045] 17 mM magnesium chloride

[0046] 90 mM potassium chloride

[0047] 39U RNase inhibitor

[0048] 1 mM DTT

[0049] 0.066 μM standard RNA

[0050] 0.2 μM oligonucleotide (one of the oligonucleotides shown belowwas used)

[0051] G2-1R (Oligonucleotide complementary to base Nos.23 to 42 of SEQID No.1; SEQ ID No.2)

[0052] G2-2R (Oligonucleotide complementary to base Nos.46 to 67 of SEQID No.1; SEQ ID No.3)

[0053] G2-3R (Oligonucleotide complementary to base Nos.104 to 125 ofSEQ ID No.1; SEQ ID No.4)

[0054] G2-4R (Oligonucleotide complementary to base Nos.201 to 220 ofSEQ ID No.1; SEQ ID No.11)

[0055] G2-5R (Oligonucleotide complementary to base Nos.222 to 241 ofSEQ ID No.1; SEQ ID No.12)

[0056] G2-6R (Oligonucleotide complementary to base Nos.249 to 271 ofSEQ ID No.1; SEQ ID No.13)

[0057] G2-7R (Oligonucleotide complementary to base Nos.274 to 293 ofSEQ ID No.1; SEQ ID No.14)

[0058] G2-8R (Oligonucleotide complementary to base Nos.324 to 344 ofSEQ ID No.1; SEQ ID No.5)

[0059] G2-9R (Oligonucleotide complementary to base Nos.512 to 533 ofSEQ ID No.1; SEQ ID No.15)

[0060] G2-10R (Oligonucleotide complementary to base Nos.725 to 745 ofSEQ ID No.1; SEQ ID No.6)

[0061] G2-11R (Oligonucleotide complementary to base Nos.812 to 831 ofSEQ ID No.1; SEQ ID No.7)

[0062] G2-12R (Oligonucleotide complementary to base Nos.930 to 952 ofSEQ ID No.1; SEQ ID No.8)

[0063] G2-13R (Oligonucleotide complementary to base Nos.1061 to 1081 ofSEQ ID No.1; SEQ ID No.16)

[0064] G2-14R (Oligonucleotide complementary to base Nos.1107 to 1126 ofSEQ ID No.1; SEQ ID No.17)

[0065] G2-15R (Oligonucleotide complementary to base Nos.1222 to 1244 ofSEQ ID No.1; SEQ ID No.18)

[0066] G2-16R (oligonucleotide complementary to base Nos.1280 to 1299 ofSEQ ID No.1; SEQ ID No.19)

[0067] G2-17R (Oligonucleotide complementary to base Nos.1303 to 1322 ofSEQ ID No.1; SEQ ID No.9)

[0068] Distilled water for adjusting volume

[0069] (3) The reaction solutions were then incubated at 41° C. for 5minutes, and then 1 μl of 8 U/μl AMV-Reverse Transcriptase (Takara ShuzoCo. Ltd.; an enzyme which cleaves RNA of a double stranded-DNA/RNA) wasadded thereto.

[0070] (4) Subsequently, the PCR tubes were incubated at 41° C. for 10minutes.

[0071] (5) Modified-urea polyacrylamide gel (acrylamide concentration:6%; urea: 7M) electrophoresis was conducted to confirm the cleavedfragments after the reaction. Dyeing following the electrophoresis wascarried out with SYBR Green II™ (Takara Shuzo Co. Ltd.). Upon binding ofthe oligonucleotide to the specific site of the target RNA, RNA of thedouble stranded DNA/RNA is cleaved by the ribonuclease H activity ofAMV-Reverse Transcriptase and, thereby, a characteristic band could beobserved.

[0072] The results of the electrophoresis are shown in FIG. 1 (black andwhite inverted). If the oligonucleotide binds specifically to thestandard RNA, the standard RNA will be decomposed at this region,yielding a decomposition product having a characteristic chain length.Specific bands were confirmed with G2-1R, G2-2R, G2-3R, G2-8R, G2-10R,G2-11R, G2-12R, G2-17R. This indicated that these oligonucleotides bindstrongly to the GII type SRSV RNA under a certain condition at atemperature of 41° C. The numbers in Table 1 are assigned by designatingthe initiation base of SEQ ID No.1 in the base sequence of SEQ. ID No.10as 1. The circles in the table indicate that a specific band wasobserved, and the symbols “X” indicate that a specific band was observedtogether with a non-specific band. TABLE 1 Oligo name Position Expectedband length (base) Result G2-1R 23  91, 2799 ◯ G2-2R 46  114, 2744 ◯G2-3R 104  172, 2716 ◯ G2-4R 201  269, 2621 X G2-5R 222  290, 2600 XG2-6R 249  317, 2570 X G2-7R 274  342, 2548 X G2-8R 324  382, 2497 ◯G2-9R 512  580, 2308 X G2-10R 725  793, 2096 ◯ G2-11R 812  880, 2010 ◯G2-12R 930  998, 1889 ◯ G2-13R 1061 1129, 1760 X G2-14R 1107 1175, 1715X G2-15R 1222 1290, 1597 X G2-16R 1280 1348, 1542 X G2-17R 1303 1371,1519 ◯

Example 2

[0073] The specificities against GII type SRSV of the oligonucleotidesselected in Example 1 were confirmed.

[0074] (1) As a GI type SRSV standard RNA, an RNA comprising base Nos.1to 3861 of the structural gene of an RNA-dependent RNA polymerasederived from the base sequence of Chiba virus RNA was quantified byultraviolet absorption at 260 nm, and then diluted with an RNA diluent(10 mM Tris-HCl (pH 8.0), 0.1 mM EDTA, 1 mM DTT, 0.5 U/μl RNaseInhibitor (Takara Shuzo Co. Ltd.)) to 0.45 pmol/μl.

[0075] (2) As a GII type SRSV standard RNA, the same RNA solution as inExample 1 (SEQ ID No.10; concentration: 0.62 pmol/μl) was used.

[0076] (3) 14 μl of a reaction solution having the following compositionwas dispensed into 0.5 ml volume PCR tubes (Gene Amp Thin-WalledReaction Tube™, Perkin-Elmer Co. Ltd.)

[0077] Reaction Solution Composition (each concentration represents thatin a final reaction solution volume of 15 μl)

[0078] 60 mM Tris-HCl buffer (pH 8.6)

[0079] 17 mM magnesium chloride

[0080] 90 mM potassium chloride

[0081] 39U RNase inhibitor (Takara Shuzo Co. Ltd.)

[0082] 1 mM DTT

[0083] 0.066 μM standard RNA

[0084] 0.2 μM oligonucleotide (one of the oligonucleotides shown belowwas used)

[0085] G2-1R (SEQ ID No.2)

[0086] G2-2R (SEQ ID No.3)

[0087] G2-3R (SEQ ID No.4)

[0088] G2-8R (SEQ ID No.5)

[0089] G2-10R (SEQ ID No.6)

[0090] G2-11R (SEQ ID No.7)

[0091] G2-12R (SEQ ID No.8)

[0092] G2-17R (SEQ ID No.9)

[0093] (4) The above reaction solutions were then incubated at 41° C.for 5 minutes, and then 1 μl of 8 U/μl AMV-Reverse Transcriptase (TakaraShuzo Co. Ltd.) was added thereto.

[0094] (5) Subsequently, the PCR tubes were incubated at 41° C. for 10minutes.

[0095] (6) Modified-urea polyacrylamide gel (acrylamide concentration:6%, urea: 7M) electrophoresis was conducted to confirm the cleavedfragments after the reaction. Dyeing following the electrophoresis wascarried out with SYBR Green II™ (Takara Shuzo Co. Ltd.). Upon binding ofthe oligonucleotide to the specific site of the target RNA, RNA of thedouble stranded DNA/RNA is cleaved by the ribonuclease H activity ofAMV-Reverse Transcriptase and, thereby, a characteristic band could beobserved.

[0096] The results of the electrophoresis are shown in FIG. 2 (black andwhite inverted). If the oligonucleotide binds specifically to thestandard RNA, the standard RNA will be decomposed at this region,yielding a decomposition product having a characteristic chain length.The results showed that the oligonucleotides selected in Example 1 bindspecifically to GII type SRSV RNA.

[0097] As explained above, the oligonucleotides of the present inventionare oligonucleotides that complementary bind to RNA derived from GIItype SRSV, even under conditions of relatively low and constanttemperature (35-50° C., preferably 41° C.), which tend to produce anintramolecular structure in RNA and prevent binding of primers or probesthereto. Specific binding of the oligonucleotides is therefore possiblewithout heat denaturation of the target RNA. The oligonucleotides of theinvention are thus useful as oligonucleotides for cleavage,amplification, detection or the like of RNA derived from GII type SRSV,i.e. as oligonucleotide primers or oligonucleotide probes to be used inRNA amplification methods.

[0098] Furthermore, the oligonucleotides of the invention are alsouseful for amplification and detection of GII type SRSV gene.

[0099] The oligonucleotides of the invention are not limited to thesequences shown in the Sequence Listings (20 to 23 mers), and may beoligonucleotides comprising at least 10 contiguous bases within thosesequences. This is apparent from the fact that an order of 10-mer basesequence is sufficient to ensure adequate specificity of primers orprobes to target nucleic acids under relatively low temperaturecondition (preferably, at 41° C.).

Example 3

[0100] RNA amplification reactions were carried out using theoligonucleotides which specifically bind to the RNA of GII type SRSV.

[0101] (1) Of the GII type SRSV-RNA, a standard RNA (SEQ ID No.10)comprising a region of totally 2843 bases containing the entireRNA-dependent RNA polymerase gene region and a portion of the structuralprotein-coding gene region, as well as a 69 base-partial region derivedfrom the 5′ end of a vector (pCR 2.1, Invitrogen) was quantified byultraviolet absorption at 260 nm, and then diluted to 1.0×10⁴ mol/5 μlwith an RNA diluent (10 mM Tris-HCl (pH 8.0), 1 mM EDTA, 0.5 U/μl RNaseInhibitor (Takara Shuzo Co. Ltd.), 5 mM DTT). In the control testsections (negative), only the diluent was used.

[0102] (2), 20.8 μl of a solution having the following composition wasdispensed into 0.5 ml volume PCR tubes (Gene Amp This-Walled ReactionTube™, Perkin-Elmer Co. Ltd.), followed by addition of 5 μl of the aboveRNA sample.

[0103] Reaction Solution Composition (each concentration represents thatin a final reaction solution volume of 30 μl)

[0104] 60 mM Tris-HCl buffer (pH 8.6)

[0105] 17 mM magnesium chloride

[0106] 90 mM potassium chloride

[0107] 39U RNase inhibitor

[0108] 1 mM DTT

[0109] 0.25 μl of each dATP, dCTP, dGTP, dTTP

[0110] 3.6 mM ITP

[0111] 3.0 mM of each ATP, CTP, GTP, UTP

[0112] 0.16 μM first oligonucleotide

[0113] 1.0 μM second oligonucleotide

[0114] 1.0 μM third oligonucleotide

[0115] 13% DMSO

[0116] Distilled water for adjusting volume

[0117] (3) RNA amplification reactions were carried out using theoligonucleotides of the sequences listed in Table 2, as the first,second and third oligonucleotides. Solutions were prepared so that thecombinations of the first, second and third oligonucleotides would bethose as listed in Table 2.

[0118] (4) After incubating the above reaction solutions for 5 minutesat 41° C., 4.2 μl of an enzyme liquid having the following compositionwas added.

[0119] Composition of Enzyme Solution (each figure represents the amountin a final reaction solution volume of 30 μl)

[0120] 1.7% sorbitol

[0121] 3 μg bovine serum albumin

[0122] 142U T7 RNA polymerase (Gibco)

[0123] 8U AMV-Reverse Transcriptase

[0124] (Takara Shuzo Co. Ltd.)

[0125] Distilled water for adjusting volume

[0126] (5) Subsequently, the PCR tubes were incubated at 41° C. for 30minutes.

[0127] (6) In order to identify the RNA amplified portion after thereaction, agarose gel (agarose concentration 4%) electrophoresis wasperformed. Dyeing following the electrophoresis was performed with SYBRGreen II (Takara Shuzo Co. Ltd.). When an oligonucleotide probe binds tothe specific portion of the target RNA, the RNA portion between thesecond and third oligonucleotide is amplified, thereby a characteristicband could be observed.

[0128] The results of the electrophoresis are shown in FIGS. 3 to 5(black and white inverted). The chain lengths of the specific bandsamplified in this reaction are shown in Table 2. Since specific bandswere confirmed in combinations from (a) to (h), and from (k) to (l), itwas demonstrated that the oligonucleotides used in these combinationsare effective in detecting GII type SRSV. TABLE 2 Amplification producedchain length (no. of Combination 1st Oligo 2nd Oligo 3rd Oligo bases)(a) G2-1S G2-1F1 G2-8R 314 (b) G2-1S G2-1F2 G2-8R 317 (c) G2-2S G2-2F1G2-8R 289 (d) G2-2S G2-2F2 G2-8R 292 (e) G2-3S G2-3F1 G2-8R 231 (f)G2-3S G2-3F2 G2-8R 234 (g) G2-10S G2-10F1 G2-12R 219 (h) G2-10S G2-10F2G2-12R 222 (i) G2-11S G2-11F1 G2-12R 133 (j) G2-11S G2-11F2 G2-12R 136(k) G2-12S G2-12F1 G2-17R 382 (l) G2-12s G2-12F2 G2-17R 385

[0129] Table 2 shows the combinations of first, second and thirdoligonucleotides used in this example, as well as the chain lengths ofthe amplified specific bands resulted from the RNA amplificationreaction using these combinations. The 3′ end hydroxyl group of eachfirst oligonucleotide base sequence was aminated. In each secondoligonucleotide base sequence, the region of the 1st “A” to the 22nd “A”from the 5′ end corresponds to the T7 promoter region, and thesubsequent region from the 23rd “G” to the 28th “A” corresponds to theenhancer sequence. The base numbers are assigned by designating theinitiation base of the RNA-dependent RNA polymerase gene of GII SRSV inSEQ ID No.36 as 1.

[0130] First oligonucleotide

[0131] G2-1S (SEQ ID No.36, base Nos.4 to 42)

[0132] G2-2S (SEQ ID No.37, base Nos.29 to 67)

[0133] G2-3S (SEQ ID No.38, base Nos.87 to 125)

[0134] G2-10S (SEQ ID No.39, base Nos.707 to 745)

[0135] G2-11S (SEQ ID No.40, base Nos.792 to 831)

[0136] G2-12S (SEQ ID No.41, base Nos.1303 to 1322)

[0137] Second oligonucleotide

[0138] G2-1lF1 (SEQ ID No.42, base Nos.37 to 59)

[0139] G2-1F2 (SEQ ID No.43, base Nos.34 to 56)

[0140] G2-2F1 (SEQ ID No.44, base Nos.62 to 84)

[0141] G2-2F2 (SEQ ID No.45, base Nos.59 to 81)

[0142] G2-3F1 (SEQ ID No.46, base Nos.120 to 142)

[0143] G2-3F2 (SEQ ID No.47, base Nos.117 to 139)

[0144] G2-10F1 (SEQ ID No.48, base Nos.740 to 762)

[0145] G2-10F2 (SEQ ID No.49, base Nos.737 to 759)

[0146] G2-11F1 (SEQ ID No.50, base Nos.826 to 848)

[0147] G2-11F2 (SEQ ID No.51, base Nos.823 to 845)

[0148] G2-12F1 (SEQ ID No.52, base Nos.947 to 969)

[0149] G2-12F2 (SEQ ID No.53, base Nos.944 to 966)

[0150] Third oligonucleotide

[0151] G2-8R (SEQ ID No.28, base Nos.324 to 344)

[0152] G2-12R (SEQ ID No.30, base Nos.930 to 952)

[0153] G2-17R (SEQ ID No.31, base Nos.1303 to 1322)

Example 4

[0154] Combinations of oligonucleotide primers according to the presentinvention were used for specific detection of different initial copynumbers of the target GII type SRSV RNA.

[0155] (1) The same GI type SRSV standard RNA (SEQ ID No. 10) as used inExample 3 was diluted with an RNA diluent (10 mM Tris-HCl (pH 8.0), 1 mMEDTA, 0.5 U/μl RNase Inhibitor (Takara Shuzo Co. Ltd.), 5 mM DTT,) toconcentrations ranging from 1.0×10⁵ copies/5 μl to 10¹ copies/5 μl. Inthe control testing sections, only the diluent was used (Negative).

[0156] (2) 20.8 μl of a reaction solution having the composition shownbelow was dispensed into 0.5 ml volume PCR tubes (Gene Amp Thin-WalledReaction Tube™, Perkin-Elmer) followed by addition of 5 μl of the aboveRNA sample.

[0157] Reaction Solution Composition (each concentration represents thatin a final reaction solution of 30 μl)

[0158] 60 mM Tris-HCl buffer (pH 8.6)

[0159] 17 mM magnesium chloride

[0160] 150 mM potassium chloride

[0161] 39U RNase Inhibitor

[0162] 1 mM DTT

[0163] 0.25 mM each of dATP, dCTP, dGTP and dTTP

[0164] 3.6 mM ITP

[0165] 3.0 mM each of ATP, CTP, GTP and UTP

[0166] 0.16 μM first oligonucleotide (G2-1S, SEQ ID No.36, wherein its3′ end is aminated)

[0167] 1.0 μM second oligonucleotide (G2-1F2, SEQ ID No.43)

[0168] 1.0 μM third oligonucleotide (G2-8R, SEQ ID No.28)

[0169] 25 nM intercalator fluorescent pigment-labeled oligonucleotide(YO-G2 SRSV-S-G, SEQ ID No.35, labeled with an intercalator fluorescentpigment at the phosphorous atom between the 7th “T” and the 8th “A” fromthe 5′ end, and modified with a glycol group at its 3′ end hydroxyl)

[0170] 13% DMSO

[0171] Distilled water for adjusting volume

[0172] (3) After incubating the above reaction solution for 5 minutes at41° C., 4.2 μl of an enzyme solution having the following compositionand pre-incubated for 2 minutes at 41° C. was added.

[0173] Enzyme Solution Composition (each concentration represents thatin a final reaction solution of 30 μl)

[0174] 1.7% sorbitol

[0175] 3 μg bovine serum albumin

[0176] 142U T7 RNA polymerase (Gibco)

[0177] 8U AMV-Reverse Transcriptase (Takara Shuzo Co. Ltd.)

[0178] Distilled water for adjusting volume

[0179] (4) The PCR tube was then incubated at 41° C. using adirect-measurable fluorescence spectrophotometer equipped with atemperature-controller, and the reaction solution was periodic measuredat an excitation wavelength of 470 nm and a fluorescent wavelength of510 nm.

[0180]FIG. 6(A) shows the time-course changes in the fluorescenceincrease ratio (fluorescence intensity at predetermined time/backgroundfluorescence intensity) of the sample, where enzyme was added at 0minutes. FIG. 6(B) shows the relationship between the logarithm of theinitial RNA amount and the rise time (time at which the relativefluorescence reaches the negative sample's average value plus 3 standarddeviations; i.e., the time to reach 1.2). The initial RNA amount wasbetween 10¹ copies/test and 10⁵ copies/test.

[0181]FIG. 6 shows that 10³ copies were detected after approximately 20minutes. A fluorescent profile and calibration curve depending on theinitial concentration of the labeled RNA were obtained, indicating thatit is possible to quantify the GII type SRSV RNA present in unknownsamples. This demonstrated that rapid, highly sensitive detection of GIItype SRSV RNA is possible by this method.

[0182] As explained above, the present invention provides usefulcombinations of oligonucleotide primers or oligonucleotide probes whichspecifically bind to RNA derived from GII type SRSV, and rapidly amplifyand detect the target RNA, even under relatively low and constanttemperature (35-50° C. and preferably 41° C.) conditions in which an RNAin a sample would form an intramolecular structure which inhibit theprimer and probe binding.

[0183] The base lengths of the oligonucleotides in the combinations ofthe present invention are not limited to the ones concretely describedherein, and the present oligonucleotides may include those comprised ofat least 10 contiguous bases within these sequences. This is apparentfrom the fact that about 10-mer base sequence is sufficient to ensureadequate specificity of primers or probes to target nucleic acids underrelatively low temperature condition (preferably, at 41° C.).

1 53 1 1530 DNA Human calicivirus 1 ggcggtgaca ataagggaac ctactgtggtgcaccaatct taggtccagg cagtgcccca 60 aaactcagca ccaagactaa attttggagatcatccacag caccactccc acctggtacc 120 tatgaaccag cctaccttgg cggcaaggaccccagagtca agggtggtcc ttcattgcaa 180 caagttatga gggaccagct gaaaccattcactgaaccca ggggtaaacc accaaaacca 240 agtgtgttag aagctgccaa gaaaaccatcatcaatgtcc ttgaacaaac aattgatcca 300 cctcaaaagt ggtcattcgc gcaagcatgcgcatccctcg acaagaccac ctctagtggt 360 cacccgcatc acatgcggaa aaatgactgctggaacgggg agtccttcac aggcaaattg 420 gcagaccagg cttccaaggc caacctgatgtacgaagagg gaaagaacat gaccccagtt 480 tacacgggtg cgcttaagga cgagctggtcaagactgaca aaatttatgg caaaatcaaa 540 aagaggcttc tctggggctc ggacctggcgaccatgatcc ggtgcgctcg ggcttttggg 600 ggcctgatgg atgaattcaa ggcacattgtgtcacactcc ccgtcagagt gggtatgaat 660 atgaatgagg atggtcctat catctttgagagacactcca gatataaata tcactatgat 720 gctgattact ctcggtggga ctcaacacaacagagggccg tattagcagc agccttagaa 780 atcatggtta agttctcccc agaacctcatctggcccaaa aggttgcaga agaccttctc 840 tctcccagcg tgatggatgt aggtgacttcagaatatcaa tcaatgaggg tctcccctcc 900 ggggtaccct gcacctccca atggaactccatcgcccact ggctcctcac tctctgtgca 960 ctttctgagg ttacaaacct gtcccctgacattatccagg ccaactccct cttttccttc 1020 tatggtgatg atgaaattgt gagcacagacgtaaagctgg acccagagaa gttgacagca 1080 aaactcaagg aatacgggct gaaaccaacccgccctgaca agactgaggg accccttgtt 1140 atctctgagg acctgaatgg cttgaccttcctgcggagga ctgtgacccg cgatccagct 1200 ggctggtttg gaaaattgga acagagttcaatacttaggc aaatgtactg gactaggggc 1260 cctaatcatg aagacccatc tgaaacaatgataccacact cccaaagacc catacaatta 1320 atgtctttgc tgggcgaggc tgccctccacggcccagcat tctacagcaa aatcagcaag 1380 ttggtcattg cagaactaaa ggaaggtggcatggatttct acgtgcccag acaagagcca 1440 atgttcagat ggatgagatt ctcagatctgagcacgtggg agggcgatcg caatctggct 1500 cccagttttg tgaatgaaga tggcgtcgaa1530 2 20 DNA Artificial Sequence synthetic DNA 2 taagattggt gcaccacagt20 3 22 DNA Artificial Sequence synthetic DNA 3 tgagttttgg ggcactgcct gg22 4 22 DNA Artificial Sequence synthetic DNA 4 tcataggtac caggtgggag tg22 5 21 DNA Artificial Sequence synthetic DNA 5 ttgtcgaggg atgcgcatgc t21 6 21 DNA Artificial Sequence synthetic DNA 6 ttgagtccca ccgagagtaa t21 7 20 DNA Artificial Sequence synthetic DNA 7 ttctgcaacc ttttgggcca 208 23 DNA Artificial Sequence synthetic DNA 8 gagtgaggag ccagtgggcg atg23 9 20 DNA Artificial Sequence synthetic DNA 9 attaattgta tgggtctttg 2010 2910 RNA Human calicivirus 10 gcgaauuggg cccucuagau gcaugcucgagcggccgcca gugugaugga uaucugcaga 60 auucggcuug gcggugacaa uaagggaaccuacuguggug caccaaucuu agguccaggc 120 agugccccaa aacucagcac caagacuaaauuuuggagau cauccacagc accacuccca 180 ccugguaccu augaaccagc cuaccuuggcggcaaggacc ccagagucaa gggugguccu 240 ucauugcaac aaguuaugag ggaccagcugaaaccauuca cugaacccag ggguaaacca 300 ccaaaaccaa guguguuaga agcugccaagaaaaccauca ucaauguccu ugaacaaaca 360 auugauccac cucaaaagug gucauucgcgcaagcaugcg caucccucga caagaccacc 420 ucuagugguc acccgcauca caugcggaaaaaugacugcu ggaacgggga guccuucaca 480 ggcaaauugg cagaccaggc uuccaaggccaaccugaugu acgaagaggg aaagaacaug 540 accccaguuu acacgggugc gcuuaaggacgagcugguca agacugacaa aauuuauggc 600 aaaaucaaaa agaggcuucu cuggggcucggaccuggcga ccaugauccg gugcgcucgg 660 gcuuuugggg gccugaugga ugaauucaaggcacauugug ucacacuccc cgucagagug 720 gguaugaaua ugaaugagga ugguccuaucaucuuugaga gacacuccag auauaaauau 780 cacuaugaug cugauuacuc ucggugggacucaacacaac agagggccgu auuagcagca 840 gccuuagaaa ucaugguuaa guucuccccagaaccucauc uggcccaaaa gguugcagaa 900 gaccuucucu cucccagcgu gauggauguaggugacuuca gaauaucaau caaugagggu 960 cuccccuccg ggguacccug caccucccaauggaacucca ucgcccacug gcuccucacu 1020 cucugugcac uuucugaggu uacaaaccuguccccugaca uuauccaggc caacucccuc 1080 uuuuccuucu auggugauga ugaaauugugagcacagacg uaaagcugga cccagagaag 1140 uugacagcaa aacucaagga auacgggcugaaaccaaccc gcccugacaa gacugaggga 1200 ccccuuguua ucucugagga ccugaauggcuugaccuucc ugcggaggac ugugacccgc 1260 gauccagcug gcugguuugg aaaauuggaacagaguucaa uacuuaggca aauguacugg 1320 acuaggggcc cuaaucauga agacccaucugaaacaauga uaccacacuc ccaaagaccc 1380 auacaauuaa ugucuuugcu gggcgaggcugcccuccacg gcccagcauu cuacagcaaa 1440 aucagcaagu uggucauugc agaacuaaaggaagguggca uggauuucua cgugcccaga 1500 caagagccaa uguucagaug gaugagauucucagaucuga gcacguggga gggcgaucgc 1560 aaucuggcuc ccaguuuugu gaaugaagauggcgucgaau gacgccgcuc caucaaauga 1620 uggugcagcu agucucguac cagagggcauuaaugagacu augccauugg aacccguugc 1680 uggcgcaucu auugcugccc caguggcgggacaaaccaac auaauugacc ccuggauaag 1740 aacaaauuuu guacaagccc ccaauggagaguuuacagug ucaccaagaa auuccccugg 1800 agaaauuuua uuaaauuuag aauuaggaccagaucugaau ccuuauuugg cccaucuuuc 1860 aagaauguac aaugguuaug cuggagguguugaggugcaa gugcuccuug cugggaacgc 1920 guucacagca gguaagauau uguuugcagcaaucccaccu aacuuuccug uagauaugau 1980 uagcccagcu caaauuacua ugcuuccccauuugauugua gauguuagga cuuuggaacc 2040 uauuaugaua cccuugccug auguuaggaauguguucuau cauuuuaaua aucaaccuca 2100 accuagaaug agguuagugg cuaugcucuacaccccauug aggucuaaug guucaggaga 2160 ugaugucuuc acugugucuu guagaguacuaacuaggcca acuccugauu uugaauuuau 2220 uuaccuggug cccccuucug uagaguccaaaacuaaacca uucacacuac caauauuaac 2280 cauuucugaa uugaccaacu cccgguuccccauuccaauc gagcaauugu auacggcucc 2340 aaaugaaacc aauguugucc agugucagaauggcaggugc accuuagaug gagagcucca 2400 gggcacaacc cagcuguuau caagugcaguuugcucuuac aggggcagga cuguggcuaa 2460 uaauggggau aauugggacc aaaauuugcuccagcugacc uauccaaaug gugcaagcua 2520 ugaccccacu gaugaagugc cagcaccauugggcacucag gauuuuagug ggauguugua 2580 uggaguguug acccaggaca augugaaugugagcacagga gaggccaaaa augcuaaggg 2640 aauauacaua uccaccacua guggaaaauucaccccaaaa auugggucaa uuggauugca 2700 uucaauaacu gagcaugugc accccaaccaacagucgcgg uucacccccg ucggagucgc 2760 cgugaaugag aacacccccu uccagcaauggguucugcca cauuaugcag guagucucgc 2820 ucucaacacc aauuuggcac cugcuguugccccgacuuuc ccuggugagc aauugcuguu 2880 cuucaggucc cgugucccau gcguucaagg2910 11 20 DNA Artificial Sequence synthetic DNA 11 tgggttcagtgaatggtttc 20 12 20 DNA Artificial Sequence synthetic DNA 12 ttggttttggtggtttaccc 20 13 23 DNA Artificial Sequence synthetic DNA 13 tgatggttttcttggcagct tct 23 14 20 DNA Artificial Sequence synthetic DNA 14attgtttgtt caaggacatt 20 15 22 DNA Artificial Sequence synthetic DNA 15tttgccataa ttttgtcagt ct 22 16 21 DNA Artificial Sequence synthetic DNA16 ttgctgtcaa cttctctggg t 21 17 20 DNA Artificial Sequence syntheticDNA 17 cagtcttgtc agggcgggtt 20 18 23 DNA Artificial Sequence syntheticDNA 18 atttgcctaa gtattgaact ctg 23 19 20 DNA Artificial Sequencesynthetic DNA 19 gtgtggtatc attgtttcag 20 20 26 DNA Artificial sequencesynthetic DNA 20 ccaatcttag gtccaggcag tgcccc 26 21 26 DNA Artificialsequence synthetic DNA 21 caaaactcag caccaagact aaattt 26 22 26 DNAArtificial sequence synthetic DNA 22 tacctatgaa ccagcctacc ttggcg 26 2326 DNA Artificial sequence synthetic DNA 23 gggactcaac acaacagagg gccgta26 24 26 DNA Artificial sequence synthetic DNA 24 tcctcactct ctgtgcactttctgag 26 25 20 DNA Artificial sequence synthetic DNA 25 taagattggtgcaccacagt 20 26 22 DNA Artificial sequence synthetic DNA 26 tgagttttggggcactgcct gg 22 27 22 DNA Artificial sequence synthetic DNA 27tcataggtac caggtgggag tg 22 28 21 DNA Artificial sequence synthetic DNA28 ttgtcgaggg atgcgcatgc t 21 29 21 DNA Artificial sequence syntheticDNA 29 ttgagtccca ccgagagtaa t 21 30 23 DNA Artificial sequencesynthetic DNA 30 gagtgaggag ccagtgggcg atg 23 31 20 DNA Artificialsequence synthetic DNA 31 attaattgta tgggtctttg 20 32 20 DNA Artificialsequence synthetic DNA 32 agtggtgctg tggatgatct 20 33 20 DNA Artificialsequence synthetic DNA 33 ggacattgat gatggttttc 20 34 20 DNA Artificialsequence synthetic DNA 34 aattgtttgt tcaaggacat 20 35 20 DNA Artificialsequence synthetic DNA 35 ccaaggtagg ctggttcata 20 36 39 DNA Artificialsequence synthetic DNA 36 taagattggt gcaccacagt aggttccctt attgtcacc 3937 39 DNA Artificial sequence synthetic DNA 37 tgagttttgg ggcactgcctggacctaaga ttggtgcac 39 38 39 DNA Artificial sequence synthetic DNA 38tcataggtac caggtgggag tggtgctgtg gatgatctc 39 39 39 DNA Artificialsequence synthetic DNA 39 ttgagtccca ccgagagtaa tcagcatcat agtgatatt 3940 39 DNA Artificial sequence synthetic DNA 40 ttctgcaacc ttttgggccagatgaggttc tggggagaa 39 41 39 DNA Artificial sequence synthetic DNA 41gagtgaggag ccagtgggcg atggagttcc attgggagg 39 42 51 DNA Artificialsequence synthetic DNA 42 aattctaata cgactcacta tagggagaat cttaggtccaggcagtgccc c 51 43 51 DNA Artificial sequence synthetic DNA 43aattctaata cgactcacta tagggagacc aatcttaggt ccaggcagtg c 51 44 51 DNAArtificial sequence synthetic DNA 44 aattctaata cgactcacta tagggagaaactcagcacca agactaaatt t 51 45 51 DNA Artificial sequence synthetic DNA45 aattctaata cgactcacta tagggagaca aaactcagca ccaagactaa a 51 46 51 DNAArtificial sequence synthetic DNA 46 aattctaata cgactcacta tagggagactatgaaccagc ctaccttggc g 51 47 51 DNA Artificial sequence synthetic DNA47 aattctaata cgactcacta tagggagata cctatgaacc agcctacctt g 51 48 51 DNAArtificial sequence synthetic DNA 48 aattctaata cgactcacta tagggagaactcaacacaac agagggccgt a 51 49 51 DNA Artificial sequence synthetic DNA49 aattctaata cgactcacta tagggagagg gactcaacac aacagagggc c 51 50 51 DNAArtificial sequence synthetic DNA 50 aattctaata cgactcacta tagggagagcagaagacctt ctctctccca g 51 51 51 DNA Artificial sequence synthetic DNA51 aattctaata cgactcacta tagggagagt tgcagaagac cttctctctc c 51 52 51 DNAArtificial sequence synthetic DNA 52 aattctaata cgactcacta tagggagatcactctctgtg cactttctga g 51 53 51 DNA Artificial sequence synthetic DNA53 aattctaata cgactcacta tagggagatc ctcactctct gtgcactttc t 51

What is claimed is:
 1. cDNA as shown in SEQ. ID. No.1, or a fragment ora derivative thereof having a size sufficient to bind to Genogroup II(GII) type Small Round Structured Virus (SRSV).
 2. An oligonucleotidefor detection of GII type SRSV, which oligonucleotide is capable ofbinding to said GII type SRSV at specific site, and comprises at least10 contiguous bases of any of the sequences listed as SEQ. ID. Nos.2 to9.
 3. The oligonucleotide according to claim 2, wherein saidoligonucleotide is an oligonucleotide probe for cleaving said RNA atsaid specific site by binding to said specific site of said RNA.
 4. Theoligonucleotide according to claim 2, wherein said oligonucleotide is anoligonucleotide primer for DNA elongation reaction.
 5. Theoligonucleotide according to claim 2, wherein said oligonucleotide is anoligonucleotide probe a portion of which is modified or labeled with adetectable marker.
 6. The oligonucleotide according to claim 2, whereinsaid oligonucleotide is a synthetic oligonucleotide in which a portionof its base(s) is (are) modified without impairing the function of saidoligonucleotide as an oligonucleotide probe.
 7. A GII type SRSV RNAamplification process in which a specific sequence of said GII type SRSVRNA present in a sample is used as a template for synthesis of a cDNAemploying an RNA-dependent DNA polymerase, the RNA of the formed RNA/DNAhybrid is decomposed by Ribonuclease H to produce a single-stranded DNA,said single-stranded DNA is then used as a template for production of adouble-stranded DNA having a promoter sequence capable of transcribingRNA comprising said specific sequence or the sequence complementary tosaid specific sequence employing a DNA-dependent DNA polymerase, saiddouble-stranded DNA produces an RNA transcription product in thepresence of an RNA polymerase, and said RNA transcription product isthen used as a template for cDNA synthesis employing said RNA-dependentDNA polymerase, wherein said RNA amplification process beingcharacterized by employing a first primer comprising at least 10contiguous bases of any of the sequences listed as SEQ. ID. No.20 toNo.24 which has a sequence homologous to a portion of said GII type SRSVRNA to be amplified, and a second primer comprising at least 10contiguous bases of any of the sequences listed as SEQ. ID. No.25 toNo.31, which has a sequence complementary to a portion of said GII typeSRSV RNA sequence to be amplified, where either or both the first andsecond primers include the RNA polymerase promoter sequence at their 5′end.
 8. The process of claim 7, wherein said RNA amplification processis carried out in the presence of an oligonucleotide probe capable ofspecifically binding to the RNA transcription product resulting from theamplification and labeled with an intercalator fluorescent pigment, andchanges in the fluorescent properties of the reaction solution aremeasured, with the proviso that said labeled oligonucleotide isdifferent from said first oligonucleotide and said secondoligonucleotide.
 9. The detection method of claim 8, characterized inthat said probe is designed so as to complementarily bind with at leasta portion of the sequence of the RNA transcription product, and thefluorescent property changes relative to that of a situation where acomplex formation is absent.
 10. The detection method of claim 9,characterized in that said probe comprises at least 10 contiguous basesof any of the sequences listed as SEQ. ID. No.32 to No.35 or acomplementary sequence.